1. Technical Field
The present invention relates to methods and compositions for treating cancer.
2. Background Art
There is evidence that tumor cell metastasis is, in part, due to complex intercellular interactions involving adhesion and aggregation. A specific class of tumor proteins, lectins, that play a role in cell adhesion are now known to be important in tumor formation and metastasis.
Lectins are, by definition, proteins with at least one carbohydrate-binding domain. By immobilizing monosaccharides, oligosaccharides, or glycoproteins in affinity columns, lectins have been isolated from tumor tissue extracts. Generally, a tissue extract in acetone or the like is prepared to isolate the protein component from the lipid component. The acetone is then evaporated, whereupon the residue is solublized in a buffered aqueous solution. This solution is then passed through an affinity column containing the immobilized carbohydrates or glycoproteins. A number of lectins, which selectively bind to galactosides, have been isolated in this manner.
Galectin-3 is one member of the family of lectins termed galectins, formerly known as S-type or S-Lac lectins. Galectins-are classified as such due to structural similarity and characteristic affinity for xcex2-galactoside sugars (1, 2). The highest levels of galectin-3 are found in activated macrophages, basophils, mast cells, some epithelial cells, and sensory neurons. An early observation was that many tumor cells express galectins on their surface and that their expression could be involved in adhesion and invasion processes. Experimental evidence also suggested that these galectins could be cross-linked by an exogenous glycoprotein resulting in the aggregation of tumor cells. Based on these results, Raz and Lotan proposed that galectin-1 and galectin-3 could promote tumor metastasis (3). Since that time, the evidence for the role of galectin-3 in tumor adhesion, invasion and metastasis has mounted.
Galectin-3 is composed of three distinct structural motifs: a short amino terminal region of 12 amino acids, a sequence rich in G-X-Y tandem repeats characteristic of the collagen supergene family, and a carboxy-terminal half containing the globular carbohydrate recognition domain (2, 4-6). There is close homology between the galectin-3 proteins of different species, but the number of N-terminal tandem repeats differs, hence, the sizes of the proteins vary (7). The human protein is composed of 250 amino acid residues with a Mr of xcx9c31,000 (5) and a carbohydrate recognition domain extending from 117 to 250. The X-ray crystal structure of the human galectin-3 carbohydrate recognition domain complexed with lactose and N-acetyllactosamine has been published (8).
Although all galectins bind lactose with similar affinity, each galectin is more specific and has higher affinity for certain more complex saccharides (9, 10). Galectins, in general, are unusual among extracellular proteins in that they are initially mainly cytosolic but can be secreted by non-classical pathways, translocated to the cell nucleus, and endocytosed and trancytosed by cells. Galectins are thought to interact with various cell-surface and extracellular glycoproteins and glycolipids, thereby playing a role in cell adhesion, migration, and signaling. The relationship between the intra- and extracellular functions of galectins may be of great biological importance. A number of reviews of the biology of the galectins have been published (11-15).
Galectin-3 can be found on the plasma membrane, and depending on the cell type can be both the nuclear and cytoplasmic or limited to the cytoplasm (6, 16-18). Galectin-3 can be secreted and reuptaken into cells by a nonclassical mechanism (19-21). Studies of mutants of hamster galectin-3 with various deletions in the N-terminal domain have shown that the even if lacking the first 103 amino acid residues the protein is localized in the nucleus. Deletion of the first 110 amino acid residues, however, prevented nuclear localization, although the exact sequence of amino acid residues 104-110, APTGALT, was not obligatory and substitution of other unrelated sequences permitted nuclear sequestration (18). The amino acid residues 104-110 of the hamster galectin-3 protein according to the consensus sequence correspond to the amino acid residues 109-115 of the homologous human galectin-3 protein (7).
Galectin-3 shares the ability to be secreted despite the absence of a signal peptide with a number of other proteins that have unconventional intercellular transfer. These proteins are internalized by cells and are able to directly access the cytoplasm and the nucleus by a process that does not involve classical endocytosis (22). This is in contrast with the modulation of intercellular events by second messengers that bind to extracellular receptors and initiate a cascade of intracellular events that often involve transciptional regulation. Although the mechanisms for the ability of some proteins to cross biological membranes in the absence of a signal sequence are poorly understood, a number of common features have been identified. Many of the proteins can directly access the nucleus, their mechanisms for secretion often vary from their mechanisms for entry, and apolipoproteins and cholesterol can play a role (22).
Galectin-3 is isolated as a monomer but undergoes multimerization on binding to surfaces that contain glycoconjugate ligands, and the N-terminal half of the protein is required for this property (23, 24). The N-terminal domain of the protein is required for galectin-3 to have affinity for multivalent carbohydrate ligands (23, 25) and to transmit intracellular signals (6, 26). Galectin-3 promotes binding of cells to laminin and fibronectin, but the N-terminally truncated protein does not (27). Thus, the N-terminal domain appears to be necessary for the self-association of galectin-3 that are required for some of its biological functions. Galectin-3 null cells were transfected to express recombinant galectin-3 and induced tumors within 4 weeks when injected into mice. When the same galectin-3 null cells were transfected to express a mutant galectin-3 that was lacking the 11 amino terminal amino acids no tumors developed within 4 weeks (6).
A number of laboratories have studied the biology of galectin-3 that apparently is significant in cell growth, differentiation, adhesion, RNA processing, apoptosis, and malignant transformation (28). Laminin is the major non-collagenous polypeptide of basement membranes, and galectin-3 binds preferentially to mouse tumor laminin compared to human placental laminin (29). Galectin-3 has been shown to increase the binding of breast cancer cells to other extracellular matrix proteins (29, 30). In addition to increasing the binding of tumor cells to basement membranes, the interaction of cell surface galectin-3 with complementary serum glycoproteins appears to promote aggregation of tumor cells in circulation, thereby playing another important role in the pathogenesis of metastasis (31).
Expression of recombinant galectin-3 in weakly metastatic fibrosarcoma cells resulted in an increased incidence of experimental lung metastases in syngeneic and nude mice (32). In human umbilical vein endothelial cells (HUVEC) galectin-3 induces angiogenesis (33). Increased expression of galectin-3 in human colon cancer cells resulted in increased metastases, and reduction in galectin-3 expression from antisense DNA was associated with decreased liver colonization and spontaneous metastasis in athymic nude mice (34). Exogenous galectin-3 has been shown to increase invasiveness of human breast cancer cells (35), and to be a chemotactic factor for human umbilical vein endothelial cells (34). However, the endogenous expression of galectin-3 by the cells was not correlated with their invasivness (35). Introduction of human galectin-3 cDNA into the human breast cancer cells BT-549 which are galectin-3 null and non-tumorigenic in nude mice resulted in the establishment of four galectin-3 expressing clones, three of which acquired tumorigenicity when injected into nude mice (36). Nonetheless, the role of galectin-3 in cancer is complicated, and a number of different laboratories have found that decreased expression of galectin-3 is associated with increased tumorigenicity and metastasis (16, 37-39). Overall, the body of work regarding the biochemistry and function of galectin-3 provides a strong rationale for continued exploration of its therapeutic use in cancer.
Galectin-3 is not a member of the Bcl-2 family of proteins, but at residues 180-183 it contains the four amino acid motif (NWGR) conserved in the BH1 domain of the Bcl-2 family, and it has 48% sequence similarity with Bcl-2 (40). Galectin-3 has antiapoptotic activity that is abrogated by substitution of the Gly182 residue with Ala in the NWGR motif (41, 42). In T cells galectin-3 interacts with Bcl-2 in a lactose inhibitable manner and confers resistance to apoptosis induced by anti-Fas antibody and staurosporine (40). Galectin-3 has been found to improve cellular adhesion and prevent apoptosis induced by loss of cell anchorage (anoikis) (42-44). Contact with the extracellular matrix is required for suppression of apoptosis of epithelial cells from a number of tissues.
By providing a mechanism for adherence of tumor cells to one another and to the extracellular matrix (27) and the subsequent suppression of apoptotis, galectin-3 on the surface of tumor cells appears to contribute to tumor invasion and metastasis. This premise is supported by the inhibition of spontaneous metastasis in a rat prostate cancer model by oral administration of modified citrus pectin, a complex polysaccharide rich in galactosyl residues. Citrus pectin (pH modified), a plant fiber component, can directly bind galectin-3, and can interfere with carbohydrate-mediated cell-cell and cell-matrix interactions (45).
U.S. Pat. No. 5, 681,923, to Platt (1997), discloses the active site of galactose binding proteins. Two different peptide sequences are claimed that correspond to part of galectin-3 are claimed. One peptide is composed of 25 amino acids corresponding to residues 171 to 196 of galectin-3. The second peptide is 38 amino acids and corresponds to residues 158 to 196 of galectin-3. In both peptides there is a histidine instead of aspartic acid 178 of galectin-3. The X-ray crystal structure of the human galectin-3 carbohydrate recognition domain in complex with lactose and N-acetyllactosamine has been published (8). The carbohydrate recognition domain of galectin-3 extends from amino acid 117 to 250 with some of the residues between 144 and 184 directly involved in hydrogen binding to lactosaminylated substrates (8). It would instead be useful to develop a larger peptide region for use in treating cancer. A larger fragment would be likely to be more effective than the peptides that were disclosed in the Platt patent.
U.S. Pat. No. 5,801,002, to Raz (1998), discloses sequences for the human galectin-3 protein. However, there is not disclosure regarding use of inhibition of the multimerization of galectin-3 that is required for many of the biological functions of the protein in cellular adhesion and signaling as a treatment. The N-terminal domain of the protein is critical for the multimerization of galectin-3 when it is bound to carbohydrate ligands. Thus, it could be useful to develop a fragment of galectin-3 having a deleted the N-terminal region so that it would still have the carbohydrate recognition domain and would still be able to bind to carbohydrate ligands for use in treating patients. This is because the N-terminally truncated galectin-3 does not have the ability to cross-link cells with other cells and to extracellular matrix. U.S. Pat. No. 5,801,002 describes using peptides as therapeutic agents that correspond to at least 4 consecutive amino acid residues of galectin-3. However, in U.S. Pat. No. 5,801,002 there is no indication that one or another of these would be better or worse in inhibition of metastasis.
U.S. Pat. 5,895,784, to Raz et al. (1999), discloses the use of pH-modified citrus pectin to treat cancer. Additionally, the Raz et al. patent describes the function, structure and expression of galectin-3. The inventors indicate that they used citrus pectin to study the properties of galectin-3 and claim a method of treating cancer by oral administration of modified pectin that can bind to the carbohydrate domain of galectin-3 to reduce metastasis. There is no disclosure of a truncated form of galectin-3 that prevents tumorigenicity and metastasis.
PCT WO 98/122139 (PCT/US97/21807), to Huflejt et al. (1998), discloses the detection of human galectin-4 in various samples. Although the focus is on galectin-4, some of the methods described include the use of galectin-3 as controls. Again, there is no disclosure of a truncated form of galectin-3 that prevents tumorigenicity and metastasis is not disclosed nor described.
U.S. Pat. No. 5, 837,493, to Hillman et al. (1998), discloses some descriptions of galectin-1, -2, and -3 including descriptions of the lectin structures and amino acid sequences. The sequence and the prevention of disease by two novel human galectins are disclosed. However, there is no disclosure of a truncated form of galectin-3 that prevents tumorigenicity and metastasis is not disclosed nor described.
The regulatory mechanism that produces the variable localization of galectin-3 in different cell types is not understood, and the significance of the relative amounts of the protein found in the cytoplasm, or nucleus, or extracellular matrix of various cell types in terms of functionality is not understood. Many laboratories have studied the role of galectin-3 in cancer, and although the results of the studies are somewhat confusing they do indicate that galectin-3 is of significance in some types of cancer. However, the prior art is lacking in a methodology and a composition of galectin-3 that can be successfully used to treat cancer. It would therefore be useful to develop a method and a composition based on an N-terminally truncated form of galectin-3 that can be used successfully to reduce tumor growth and metastasis.
According to the present invention, there is provided a composition having an effective amount of an N-terminally truncated galectin-3 that is lacking the N-terminal 107 amino acids in a pharmaceutically acceptable carrier. Also provided by the present invention is a method of treating a cancer in a patient by administering to a patient in need of such treatment an effective amount of N-terminally truncated galectin-3 in a pharmaceutically acceptable carrier.